Proposal form for the evaluation of a genetic test for NHS Service Gene Dossier/Additional Provider TEST DISEASE/CONDITION POPULATION TRIAD Submitting laboratory: London North East RGC GOSH Approved: September 2012 1. Disease/condition approved name and Lymphoproliferative Syndrome 1; LPFS1 symbol as published on the OMIM database (alternative names will be listed on the UKGTN website) 2. OMIM number for disease/condition 613011 3. Disease/condition please provide a brief description of the characteristics of the disease/condition and prognosis for affected individuals. Please provide this information in laymen s terms. 4. Disease/condition mode of inheritance Autosomal recessive 5. Gene approved name(s) and symbol as published on HUGO database (alternative names will be listed on the UKGTN website) EBV-associated autosomal lymphoproliferative syndrome (ITK deficiency) presents with a similar but not identical phenotype to X-linked lymphoproliferative disease (XLP) characterised by lymphoproliferation and severe immune dysregulation following Epstein- Barr virus (EBV infection) and is inherited in an autosomal recessive manner. As in XLP, not all 3 classic clinical manifestations (fulminant infectious mononucleosis, lymphoproliferative disorder and dysgammaglobulinaemia) may develop and patients can develop different immune mediated syndromes including fatal haemophagocytosis, hypogammaglobulinaemia and autoimmune phenomena presenting as renal disease. While in XLP the predominant lymphoproliferation is Burkitt s lymphoma, 4 out of 5 published patients with ITK deficiency had Hodgkin s disease. Unlike XLP, infection with EBV is a universal component of the phenotype. Most affected individuals will die in childhood following EBV infection unless treated successfully by bone marrow transplantation (BMT) or by cord-blood stem cell transplant. Regular immunoglobulins are used for hypogammaglobulinaemia. IL2-inducible T-cell kinase; ITK 6. OMIM number for gene(s) 186973 7. Gene description(s) The IL2-inducible T-cell kinase (ITK) gene is located at 5q33.3 and consists of 17 exons. ITK is a member of the Tec Kinase family. These proteins are important mediators of antigen receptor signalling in lymphocytes, and ITK is believed to be the predominant Tec kinase in T cells and one of the key molecules involved in NKT cell maturation and survival (1). ITK is the T-cell-specific homolog of Bruton tyrosine kinase (BTK), which is mutant in X-linked agammaglobulinemia. BTK interacts with 3 protein-
7b. Number of amplicons to provide this test 7c. MolU/Cyto band that this test is assigned to tyrosine kinases that get activated upon stimulation of B- and T-cell receptors. These interactions are mediated by two 10-amino acid motifs in BTK; an analogous site with the same specificity is also present in ITK (2). (1) Stepensky et al Il-2-inducible T-cell kinase deficiency: clinical presentation and therapeutic approach. Haematologia 2011: 96(3) (2) Cheng et al Binding of Bruton's tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domainmediated interaction. Proc. Nat. Acad. Sci. 91: 8152-8155, 1994. 17 MolU Band D 2012/13 MolU Band E 2013/14 8. Mutational spectrum for which you test Full screen for point mutations and small deletions including details of known common and duplications in ITK by direct Sanger sequence mutations analysis. No common mutations have been reported. 9. Technical method(s) Bi-directional Sanger sequence analysis of ITK 10. Validation process Please explain how this test has been validated for use in your laboratory Sequencing primers have been designed, SNP checked and optimised for all amplicons. 10 normal controls and 14 patient samples have been screened by sequence analysis. The laboratory also participates in all relevant technical EQA schemes that are available through UK NEQAS and EMQN: 2011 In progress 2007 -Genotypes correctly assigned in 3/3 sequencing EQA samples 2006 - Genotypes correctly assigned in 3/3 sequencing EQA samples 2005 - Genotypes correctly assigned in 3/3 sequencing EQA samples 2004 - Genotypes correctly assigned in 4/4 sequencing EQA samples. 11a. Are you providing this test already? Yes 11b. If yes, how many reports have you produced? 11c. Number of reports mutation positive 0 11d. Number of reports mutation negative 14 12. For how long have you been providing this service? 13a. Is there specialised local clinical/research expertise for this disease? 14 2 months Yes
13b. If yes, please provide details Dr Catherine Cale, Clinical Immunology, Great Ormond Street Hospital, London 14. Are you testing for other genes/diseases/conditions closely allied to this one? Please give details EBV-associated autosomal lymphoproliferative syndrome (ITK deficiency) presents with a similar but not identical phenotype to X-linked lymphoproliferative disease (XLP). We currently offer testing for XLP1 (SH2D1A gene) and XLP2 (XIAP gene). We also test for a number of other immunodeficiency syndromes, including Wiskott-Aldrich, severe combined immunodeficiency (SCID), X-linked Hyper IGM, Cartilage hair hypoplasia and X-linked agammaglobulinaemia as part of the immunodeficiency service repertoire. Your current activity If applicable - How many tests do you currently provide annually in your laboratory? 15a. Index cases 10 15b. Family members where mutation is known Your capacity if Gene Dossier approved How many tests will you be able to provide annually in your laboratory if this gene dossier is approved and recommended for NHS funding? 16a. Index cases 20 16b. Family members where mutation is 40 known Based on experience how many tests will be required nationally (UK wide) per annum? Please identify the information on which this is based 17a. Index cases 10 17b. Family members where mutation is known 18. National activity (England, Scotland, Wales & Northern Ireland) If your laboratory is unable to provide the full national need please could you provide information on how the national requirement may be met. For example, are you aware of any other labs (UKGTN members or otherwise) offering this test to NHS patients on a local area basis only? This question has been included in order to gauge if there could be any issues in equity of access for NHS patients. It is appreciated that some laboratories may not be able to answer this question. If this is the case please write unknown. As no index cases with a mutation have been identified to date, family mutation testing has so far not been necessary 4 This laboratory will be able to provide a national service
EPIDEMIOLOGY 19. Estimated prevalence of condition in the general UK population Please identify the information on which this is based Rare The first molecular cause of an autosomal recessively inherited lymphoproliferative disease was published in 2009 (2 girls from a consanguineous Turkish family) (1) and a further paper in 2011 described 3 affected individuals from a family of Arab origin (2) 1. Huck K et al Girls homozygous for an IL-2 inducible T cell kinase mutation that leads to protein deficiency develop fatal EBV-associated lymphoproliferation, 2009, J Clin Invest. 119, 1350 2. Stepensky et al Il-2-inducible T-cell kinase deficiency: clinical presentation and therapeutic approach. Haematologia 2011: 96(3) 20. Estimated gene frequency (Carrier frequency or allele frequency) Unknown Please identify the information on which this is based 21. Estimated penetrance Please identify the information on which this is based Fully penetrant with a variable phenotype. 4 out of 5 published patients with ITK deficiency had Hodgkin s disease (1) 1. Stepensky et al Il-2-inducible T-cell kinase deficiency: clinical presentation and therapeutic approach. Haematologia 2011: 96(3) 22. Estimated prevalence of condition in the target population. The target population is the group of people that meet the minimum criteria as listed in the Testing Criteria. Unknown - the association between ITK mutations and EBV-associated lymphoproliferation has only recently been elucidated. INTENDED USE 23. Please tick the relevant clinical purpose of testing Diagnosis Yes No Treatment Yes No Prognosis & management Yes No Presymptomatic testing Yes No Carrier testing for family members Yes No Prenatal testing Yes No
TEST CHARACTERISTICS 24. Analytical sensitivity and specificity This should be based on your own laboratory data for the specific test being applied for or the analytical sensitivity and specificity of the method/technique to be used in the case of a test yet to be set up. Direct Sanger sequencing has a high sensitivity in this laboratory. We use Big Dye chemistry, ABI analysers (3130XL, 3730XL) and analyse using Mutation Surveyor software. We participate and perform successfully in EQA programmes for sequence analysis (see above). Bidirectional sequence analysis has specificity approaching 100% although large insertions/deletions and deep intronic mutations will not be detected. 25. Clinical sensitivity and specificity of test in target population The clinical sensitivity of a test is the probability of a positive test result when condition is known to be present; the clinical specificity is the probability of a negative test result when disease is known to be absent. The denominator in this case is the number with the disease (for sensitivity) or the number without condition (for specificity). Clinical sensitivity and specificity is expected to be high, although variants of uncertain pathogenicity may be identified during testing of affected or unaffected individuals. Given the rarity of this condition and significant clinical overlap with other genetic diseases, it is difficult to determine sensitivity and specificity of this assay. 26. Clinical validity (positive and negative predictive value in the target population) The clinical validity of a genetic test is a measure of how well the test predicts the presence or absence of the phenotype, clinical condition or predisposition. It is measured by its positive predictive value (the probability of getting the condition given a positive test) and negative predictive value (the probability of not getting the condition given a negative test). The positive predictive value is high, patients with biallelic mutations are expected to manifest EBVassociated autosomal lymphoproliferative syndrome (ITK deficiency), although phenotype is variable. Similarly the NPV is high; absence of biallelic ITK mutations indicates the patient is not affected with EBV-associated autosomal lymphoproliferative syndrome (ITK deficiency). However an alternative cause for the patient phenotype has not been ruled out and is likely to be due to mutation in an unidentified gene. 27. Testing pathway for tests where more than one gene is to be tested Please include your testing strategy if more than one gene will be tested and data on the expected proportions of positive results for each part of the process. Please illustrate this with a flow diagram. Haemophagocytic lymphohistiocytosis (HLH) with Epstein Barr virus (EBV): Female: investigate for ITK deficiency by protein expression analysis and molecular mutation screen in parallel. Male: investigate for XLP1, XLP2 by protein expression analysis followed by mutation screening if indicated. Test for ITK deficiency by protein expression analysis and molecular mutation screening in parallel. CLINICAL UTILITY 28. How will the test add to the management of the patient or alter clinical outcome? A patient with ITK deficiency confirmed by mutation analysis would progress to bone marrow transplantation (HSCT). Without this information HSCT may be delayed or not performed in a timely manner resulting in the patient s death. A successful bone marrow transplantation has been reported in one patient with EBV-associated autosomal lymphoproliferative syndrome (1) (1) Stepensky et al Il-2-inducible T-cell kinase deficiency: clinical presentation and therapeutic approach. Haematologia 2011: 96(3) 29. How will the availability of this test impact on patient and family life? A confirmed diagnosis of ITK deficiency allows prenatal diagnosis for at risk couples and testing of relatives. Diagnosis also allows clarification of recurrence risk and appropriate genetic counselling Definitive diagnosis enables full information to be given to families regarding extent of the disease; appropriate treatment can also be initiated as soon as possible.
30. Benefits of the test Please provide a summary of the overall benefits of this test. Further genetic testing will be unnecessary if pathogenic mutations in ITK are identified as the cause of a patient s phenotype. Appropriate clinical management can be implemented early if a definitive diagnosis is made. Depending on the presentation, appropriate management may be HLH2004 or lymphoma treatment followed in both cases by HSCT. In the absence of a confirmed diagnosis additional unnecessary investigation may be undertaken, for example genetic screening for other HLH and EBV disease associated genes (7 genes), further infectious disease investigations (e.g. leshmanias) and investigation for primary immunodeficiency (e.g. lymphocyte subsets, proliferation assays) and there may be delay in appropriate clinical management of the patient. Affected family members may be identified, supporting very early and pre-symptomatic intervention. 31. Is there an alternative means of diagnosis or prediction that does not involve molecular diagnosis? If so (and in particular if there is a biochemical test), please state the added advantage of the molecular test. ITK protein expression is screened by the Molecular Immunology laboratory at GOSH by Western blot (immunoblot) in a recently developed test. However, as yet no patient with confirmed EBV-associated autosomal lymphoproliferative syndrome (ITK deficiency) has been identified and the protein test and molecular test need to run in parallel for all patients until the specificity and sensitivity of the protein test has been established. Following this, the protein test will be used as pre-screen and only patients with ITK deficiency or an inconclusive result will proceed to molecular testing. 32. Please describe any specific ethical, legal or social issues with this particular test. None 33. The Testing Criteria must be completed where Testing Criteria are not already available. If Testing Criteria are available, do you agree with them Yes/No If No: Please propose alternative Testing Criteria AND please explain here the reasons for the changes. 34. Savings or investment per annum in the diagnostic pathway based on national expected activity, cost of diagnostics avoided and cost of genetic test. Please show calculations. Based on costs below, and expected national activity of 10 referrals for ITK deficiency the cost of molecular test would be: 10 x 450 = 4500 Saving of approximately 500 on other pathology tests per case 10 x 500 = 5000 Annual savings 5000-4500 = 500 35. List the diagnostic tests/procedures that would no longer be required with costs. Costs and type of imaging procedures Costs and types of laboratory pathology tests (other than molecular/cyto genetic proposed in this gene dossier) Costs and types of physiological tests (e.g. ECG) Cost and types of other investigations/procedures (e.g. biopsy) Total cost tests/procedures no longer required 500 Repeated & varied testing that is patient specific (e.g. ferritins; scd25 ) 500
36. REAL LIFE CASE STUDY In collaboration with the clinical lead, describe a real case example to illustrate how the test would improve patient experience No mutation positive cases have yet been described at Great Ormond Street so here we have summarised the first published report of clinical cases that led to the identification of ITK. Huck K et al Girls homozygous for an IL-2 inducible T cell kinase mutation that leads to protein deficiency develop fatal EBV-associated lymphoproliferation, 2009, J Clin Invest. 119, 1350 In the first report of ITK deficiency a consanguineous Turkish family are described in which 2 sisters died after showing fatal EBV-associated autosomal lymphoproliferative disorder that clinically resembled XLP. The older sister presented at 6 years of age and an extensive immunological work-up failed to determine the underlying defect. There were several recurrences of EBV infection and an EBVassociated Hodgkin lymphoma was also identified in the following three years. At 10 years and 5 months the child succumbed to infection and died of respiratory failure. Her younger sister showed no signs of being similarly affected at 16 months of age, but the condition could not be excluded. She presented with recurrent episodes of fever at age 5 that was shown 4 months later to be caused by an active EBV infection. After multi-organ failure and attempted stem cell transplantation the girl died aged 5 years and 9 months. Due to the consanguinity and similar presentation of the sisters, genome wide association studies and candidate gene screening led to the identification of a homozygous ITK mutation: c.1085c>t (p.arg335trp) that was not detected in 200 controls and caused nearly undetectable ITK protein expression. Had the molecular defect been elucidated sooner in this family, molecular testing may have identified the ITK deficiency in the first sibling enabling bone marrow transplantation. The younger sister could then have had pre-symptomatic testing at 16 months when still apparently healthy enabling early treatment. Parents of the sisters may also now be offered prenatal diagnosis. 37. For the case example, if there are cost savings, please provide these below: Costs are not provided because the case study has been drawn from a publication and not from the genetic service. PRE GENETIC TEST Costs and type of imaging procedures Costs and type of laboratory pathology tests (other than molecular/cyto genetic proposed in this gene dossier) Costs and type of physiological tests (e.g. ECG) Cost and type of other investigations/procedures (e.g. biopsy) Cost outpatient consultations (genetics and non genetics) Total cost pre genetic test POST GENETIC TEST Costs and type of imaging procedures Costs and types laboratory pathology tests (other than molecular/cyto genetic proposed in this gene dossier) Cost of genetic test proposing in this gene dossier Costs and type of physiological tests (e.g. ECG) Cost and type of other investigations/procedures (e.g. biopsy) Cost outpatient consultations (genetics and non genetics) Total cost post genetic test 38. Estimated savings for case example described
UKGTN Testing Criteria Approved name and symbol of disease/condition(s): Lymphoproliferative Syndrome 1; LPFS1 OMIM number(s): 613011 Approved name and symbol of gene(s): IL2-inducible T-cell kinase; ITK OMIM number(s): 186973 Patient name: Patient postcode: Date of birth: NHS number: Name of referrer: Title/Position: Lab ID: Referrals will only be accepted from one of the following: Referrer Consultant Immunologists Consultant Clinical Geneticists Tick if this refers to you. Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria Epstein-Barr virus (EBV) infection AND Haemophagocytic lymphohistiocytosis (HLH) OR Hodgkin lymphoma AND ITK immunoblot performed by GOSH Immunology AND XLP1 (SH2D1A ) and XLP2 (XIAP) mutations have been excluded (males only) OR At risk family members where familial mutation is known Tick if this patient meets criteria If the sample does not fulfil the clinical criteria or you are not one of the specified types of referrer and you still feel that testing should be performed please contact the laboratory to discuss testing of the sample